【作者】 张宁；

【导师】 余丽芸；

【作者基本信息】 黑龙江八一农垦大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 由于大部分病原体是由黏膜进入机体,黏膜免疫对机体防御病原体的入侵有着十分重要的作用。为了避免传统注射疫苗的缺点,人们正在研制开发一种新型的黏膜疫苗。黏膜疫苗价格相对低廉且能够诱导机体产生局部和系统免疫应答,可以有效地阻止病原体入侵机体。乳酸菌是一种安全的微生物,可作为口服疫苗活载体将抗原传递到黏膜表面。本研究将PCR扩增的绿色荧光蛋白(Green Fluorescent Protein,GFP)基因分别克隆到可在大肠杆菌和乳酸菌中表达的穿梭质粒载体pLA和pLA-A上,构建出重组质粒pLA-GFP和pLA-A-GFP,电转化至Lactobacillus casei(L.casei)中,利用多聚谷氨酸跨膜蛋白pgsA基因锚定在细胞表面,使得外源抗原获得稳定高效的表达。经过SDS-PAGE、Western blot检测,约有69 KDa和79 KDa的融合蛋白得到表达,蛋白大小与理论值相符合。荧光观察和流式细胞术的结果表明,pgsA成功的将融合蛋白展示在菌体表面。将重组菌pLA-GFP/L.casei.口服免疫SPF级BALB/c小鼠,重组菌能在肠道黏膜的不同部位以一定的比例存活并附着在肠黏膜表面,口服6 d后达到定植高峰期。7 d后,在十二指肠、空肠、回肠和盲肠定植率分别占第1天的16.49%、25.08%、47.71%、41.03%。研究结果为口服免疫程序的制定奠定了基础,并为其提供了具有一定参考价值的试验数据。将重组菌pLA-A-GFP/L.casei口服免疫SPF级BALB/c小鼠,免疫后不同时间采集血液样品、肺部、肠道冲洗液及粪便样品,利用间接ELISA方法测定小鼠血清中IgG抗体水平和肺部、肠道冲洗液及粪便样品中特异性sIgA抗体水平的动态规律。结果表明重组pLA-A-GFP/L.casei口服免疫小鼠后能够产生特异性抗体,不但检测到高水平血清IgG,而且在肠道冲洗液及粪便样品均可产生sIgA抗体,连续口服免疫5天所产生的抗体水平高于其它试验组。该结果证明了重组菌pLA-A-GFP/L.casei可以刺激动物机体产生系统免疫应答和黏膜免疫应答,为动物免疫机理的进一步研究和乳酸菌黏膜疫苗的研制奠定了基础。更多还原

【Abstract】 As many parasitic pathogens enter the body via mucosal surfaces, mucosa immunization was very important to protect organisms from infection of these pathogens. New vaccination strategies have been developed in order to avoid disadvantages associated with parenterally administeration vaccines. Oral vaccination applied on a large scale,is relatively inexpensive and frequently induces both local and systemic immune responses, resulting in an effective blocking the route of pathogens in entry and infection site. Lactic acid bacteria (LAB) are considered to be safe organisms and advocated for use as live antigen delivery vehicles to mucosal sites.In this study, Green Fluorescent Protein (GFP) was cloned into the expression vectors pLA and pLA-A, which can shuttle and express in E.coli and Lactobacillus casei (L.casei), and transformed into the host cells L.casei by electroporation to generate recombinant becteria pLA-GFP/ L.casei. and pLA-A-GFP/ L.casei. The recombinant fusion proteins were stable expressed on cell surface using the poly-r-glutamic acid synthetase A protein (pgsA) as an anchoring matrix. The molecular weight of the recombinant protein was about 69 kDa and 79 kDa in the result of Western blot. The GFP fusion protein on the cell surface was confirmed by fluorescence microscopy and flow cytometric analysis. We orally dosed up six-week-old female SPF BALB/c mice with the recombinant L.casei of approximately 109. Groups of at least three mice per condition were sacrificed at 1.5 h, 3 h, 12 h, 1 d, 3 d, 5 d, 6 d, 7 d, and its duodenum, jejunum, ileum, caecum intestinal tract rinse solution was sampled separately. The recombinant bacteria in intestinal tracts were examined by the method of plate culture count. The ratio of the seventh day to the first day of the recombinant L.casei adhered to the intestinal mucosa in the duodenum, jejunum, ileum, and caecum was 16.49%, 25.08%, 47.71%, and 41.03%, respectively. Our findings indicated that L.casei used as a deliver vector in oral vaccine is feasible.Oral immunization of SPF BALB/c mice was performed with the recombinant strain L.casei harboring pLA-A-GFP. Specific anti-TGEV IgG antibody in the serum and specific anti-TGEV secret immunoglobulin A (sIgA) antibody in the lung intestines fluid and feces of mice were detected by indirect ELISA. The results showed that the mice immunized with recombinant bacteria produced high level of anti-TEGV IgG in the serum and high level of sIgA antibodies were detected in the vaccinated group. The results of five consecutive days oral immunization group have higher level of antibody than others. These results indicated that the recombinant pLA-A-GFP/L.casei could induce serum IgG responses as well as specific mucosal IgA responses, and evoked both system immune and mucosal immune responses against TGEV.更多还原